This proposal concerns a biochemical investigation of mechanisms involved in the transcription of specific genes during cell growth and differentiation and in malignant cells. The major emphasis will be on specific 5S RNa, tRNA, and histone genes expressed in Xenopus laevis oocytes and embryos, in insect tissues and in mammalian cells (mouse plasmacytoma and human KB). The general approach involves the transcription of these genes, by homologous and heterologous eukaryotic RNA polymerases (I, II, and III), in reconstituted systems. Previous studies in this lab have demonstrated the accurate transcription of class III genes (by RNA polymerase III) in chromatin templates or in purified DNA templates in the presence of other soluble factors. Further, studies of class III genes will be directed toward 1) the purification of the specificity factors thus far identified; 2) an analysis of their mechanism of action, e.g., whether they act at termination or initiation and whether primary interactions are with the template or the RNA polymerase; 3) an analysis of the effects of chromatin structure upon the transcription of purified genes by these factors, 4) a search for other (gene specific) factors which may be necessary to activate these genes within a chromatin structure; and 5) the cloning and analysis of additional class III genes, e.g., those encoding specific tRNAs, from the various species. In addition, attempts will be made to clone class II genes (those encoding mRNAs and transcribed by RNA polymerase II) and to analyze their transcription in cell free systems as outlined for the class III genes.